Abstract
Abstract Background: ERCC1 is a DNA repair protein whose expression is a prognostic and predictive biomarker of chemotherapy effect in NSCLC. Four alternative transcripts of the ERCC1 gene have been described (isoforms 201 to 204). We previously found that only ERCC1-202 isoform is able to remove cisplatin-DNA adducts and to improve survival after cisplatin treatment. Little is known however about the role of the other isoforms. Since the four isoforms are expressed in human samples, we sought to elucidate the contribution of these different ERCC1-isoforms to ERCC1 described functions within and beyond DNA repair. Methods: We used an established ERCC1-deficient A549 cells with selective re-expression of each ERCC1 isoform. We searched for negative dominant functions of the ERCC1-201, -203, and -204 isoforms using proliferation assay (WST-1) and clonogenic assay. Respective cellular localizations of the isoforms were assessed by immunofluorescent (IF) staining and interacting abilities with previously identified ERCC1 partners was estimated by proximity ligation assays (Duolink). We finally investigated their influence on the cellular mitotic process (mitotic spindle shape) by alpha- and gamma-tubulin IF and video microscopy. Results: IF detection of ERCC1 protein isoforms revealed a nuclear localization of ERCC1-201 and -202 isoforms whereas ERCC1-203 and -204 isoforms were mainly detected in the cytoplasm. This alternative cellular localization suggests alternative roles for ERCC1-203 and -204. Only ERCC1-202 formed a stable heterodimer complex with XPF and other interacting partners (XPA, MSH2, FANCG, SLX4 and TRF2). None of the isoforms decreased cisplatin resistance (IC50) associated with ERCC1-202 expression. Interstrand Cross-Link repair (ICL-R) efficiency (mitomycin-C treatment) was also exclusively rescued with ERCC1-202 re-expression. Further, ERCC1 deficient cells displayed mitotic aberrations such as chromosome bridges at anaphase that impaired cytokinesis and generated aneuploidy. Only ERCC1-202 isoform restored chromosome segregation accuracy. Conclusions: Alternative roles for ERCC1 beyond the canonical NER pathway are currently emerging. Our data suggest that all currently known functions of ERCC1 are fulfilled by the same ERCC1 isoform ERCC1-202. Despite their wide expression in human samples, the identification of alternative roles of other isoforms is still under investigation. Our A549 clones, with stable ERCC1 deficiency, provide a promising tool to analyze different functions of ERCC1. Finally, most antibodies used to test ERCC1 status in patients do not distinguish between different isoforms, it is important to develop an antibody (or alternative diagnostic method) able to specifically recognize only the functional ERCC1-202 isoform. Given that ERCC1-202/XPF interaction domains can be produced together it would be realistic to use it as an immunogenic epitope and/or a therapeutic target. Citation Format: Luc Friboulet, Ken André Olaussen, Florence Ponsonnailles, Annabelle Stoclin, Nicolas Dorvault, Julien Adam, Frédéric Commo, Patrick Saulnier, Fabrice André, Jean-Charles Soria. ERCC1-202 isoform is responsible for all known functions of ERCC1. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 630. doi:10.1158/1538-7445.AM2013-630
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